云南越冬红嘴鸥弓形虫的生物学特性研究

Wintering Black-Headed Gulls have been nice landscape in Yunnan.Our previous investigation indicates that Toxoplasma gondii infection is common in Black-headed Gulls. It was not clear about the biological characteristics of infection source, T.gondii genotype, genetic evolution and virulence. The present study intends to collect the samples from gulls blood and tissues and stray cats, mice, raise chickensa and fish and shrimp entrails, and then detect antibodies to T. gondii using the modified agglutination test(MAT), PCR amplification B1 gene. Inoculate SPF mice using collected positive tissues or oocysts, passage HFF Cells. Genotypes will analyzed by Mn-PCR-RFLP and multilocus enzyme digestion. The introns will analyzed by MLST, and then build the genetic evolutionary tree clustering analysis from gulls and surroundings, clarify the infection sources of gulls toxoplasmosis. To measure T. gondii virulence in mice, tachyzoites inoculate mice for each dilution, record mortality, collect blood samples and determine seroconversion by MAT test, determine cumulative mortality in mice, and amplify the sequences of ROP5 and ROP18 for each strains. The results would contribute to clarify the biological characteristics after gulls infected toxoplasmosis and also provide theoretical basis for finding the source, prevention, diagnosis and treatment toxoplasmosis for migratory birds.

来云南越冬的红嘴鸥，成为了云南特色的景观。前期检测发现，红嘴鸥感染弓形虫的血清阳性率较高，但其感染来源、虫株基因型、遗传进化分支及毒力等生物学特性尚不明确。基于此，本项目拟采集红嘴鸥血和组织样及其栖息环境中流浪猫、老鼠、散养鸡和湖滩鱼虾内脏，采用MAT法检测血清抗体，巢式PCR扩增B1基因；从阳性血样、组织、环境采集物中分离的虫体/卵囊接种SPF小鼠，再传代HFF细胞；采用Mn-PCR-RFLP和多位点酶切鉴定虫株基因型、采用MLST分析各基因型虫株的内含子位点多态性，构建鸥源和环境源虫株遗传进化树进行聚类分析，阐明红嘴鸥感染弓形虫病的来源；用不同剂量的速殖子分别接种SPF小鼠，观察病症、计算累计死亡率、MAT检测抗体，以此评价虫株毒力，扩增各虫株ROP5和ROP18，测序、比对序列差异。通过上述研究，将阐明红嘴鸥所感染的弓形虫的生物学特性，为候鸟弓形虫病的溯源、预防、诊疗提供理论依据。

获悉了迁徙入云南的红嘴鸥及其栖息环境弓形虫病的流行情况，采用血清学方法检出红嘴鸥总阳性率为7.21%；采用巢式PCR扩增检出红嘴鸥血液、红嘴鸥生活周边环境猫、老鼠和散养鸡感染弓形虫阳性率为9.95%；通过遗传进化分析，发现所分离虫株与波兰水生动物、北印度的羊弓形虫在同一分支上。明确了红嘴鸥感染弓形虫的基因型及其致病性，即采用PCR-RFLP方法鉴定出红嘴鸥和红嘴鸥栖息环境中猫、老鼠和散养鸡弓形虫分离株均为ToxoDB#9（ChineseⅠ），该基因型是云南以及中国大部分地区主要流行的弓形虫基因型。采用猫源弓形虫虫株成功感染小白鼠，小白鼠表现出典型临床症状和病理变化，并分析了虫株毒力因子差异。本研究首次对云南滇池红嘴鸥及其栖息环境中部分动物弓形虫流行、虫株分子遗传和致病性进行了研究，具有重要的兽医公共卫生意义。为红嘴鸥弓形虫病的预防提供数据支撑，并补充了鸟类弓形虫生物信息库。通过该项目研究，以云南农业大学为第一作者单位、以项目编号为第一标注发表相关学术论文5篇，其中SCI收录2篇；培养硕士研究生3人；培养云南省两类人才1人，获建省级创新团队1个。